KR100397244B1 - Megakaryocyte differentiation factor - Google Patents

Abstract

PURPOSE: Provided are a megakaryocyte differentiation factor, gene coding for the factor and a process for production thereof. The megakaryocyte differentiation factor is useful as a hematopoietic stimulating factor for megakaryocyte-platelet linease. CONSTITUTION: A megakaryocyte differentiation factor has the following properties; (1) stimulating differentiation of megakaryocytes; (2) exhibiting a molecular weight of 55 to 57 kD as determined by gel filtration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and having no intermolecular disulfide linkage; (3) exhibiting an isoelectric point of 6.5+-0.5; and (4) having at least one of the amino acid sequences shown in SEQ ID NO: 1 to 9.

Description

Megakaryocyte differentiation factor

The present invention relates to a megakaryocyte differentiation factor, a gene encoding the factor, and a method for producing the same. Megakaryocyte differentiation factor is useful as a hematopoietic stimulating factor in megakaryocyte platelet lineage.

It is well known that various hematopoietic factors that induce the growth and differentiation of blood cells are involved in the process of maturing hematopoietic stem cells into blood cells.

Although the life cycle of platelets is short, from 9 to 10 days, the concentration of platelets in the blood remains rather constant during the resting period. Moreover, even if the number of platelets is artificially reduced by one of a variety of useful methods in laboratory animals, the number of platelets in the blood recovers within a few days. From these facts, it is speculated that there is a factor that stimulates platelet formation and much effort has been made to identify this factor.

It is contemplated that two or more regulatory factors will be included in the megakaryocyte platelet hematopoietic lineage. The first factor is called a megakaryocyte colony stimulating factor because it stimulates the formation of megakaryocyte colonies by itself.

The second factor is not itself active to stimulate the formation of megakaryocyte colonies, but is called a megakaryocyte potentiator because it binds to the first factor to increase the number and stimulate differentiation of the megakaryocyte colonies.

The megakaryocyte colony stimulating factor includes interleukin 3, and granulocyte / macrophage colony stimulating factor, and the megakaryocyte potentiator includes erythropoietin, macrophage colony stimulating factor, interleukin 6,7 and 11, LIF, and the like. Some of these factors have the effect of increasing the number of platelets in vivo or shortening the time required to recover the number of platelets (see Hideaki Mizoguchi: Tanpakushitsu kakusan Koso 36, 1195, 1991, Japan).

However, most of these factors exhibit a variety of biological activities besides being involved in the differentiation of blood cells in various hematopoietic lineages, including differentiation in megakaryocyte-platelet lineages. For example, although IL-6 and IL-11 actually exhibit progenitor action in vivo, they stimulate the production of acute phase proteins and, in severe cases, cachexia. Moreover, IL-6 is associated with a variety of clinical problems; For example, IL-6 can stimulate the growth of vasculature cells in the kidney, resulting in renal failure (see Tadashi Matsuda et al ,, Tanpakushitsu Kakusan Koso, 36, 1184, 1991, Japan), moreover, IL -6 is not considered as a physiological factor because it does not exhibit high blood concentrations during the platelet reduction phase.

Platelets play an important role in the hemostatic mechanism. Diseases associated with thrombocytopenia (Fancony syndrome, megakaryocytic thrombocytopenia, aplastic anemia, etc.) are clinically dangerous and, in particular, bleeding cannot be controlled. Thus, the isolation and identification of factors that stimulate the production of platelets is considered to be useful in preventing the aforementioned risks.

Currently, bone marrow transplantation has become a powerful treatment for treating leukemia, and the success rate has been increased by using cytokines such as erythropoietin (EPO) and granulocyte colony stimulating factor (G-CSF). The current problem with bone marrow transplantation is the reduction in platelet count, and if the progenitor factor is available, it is expected that the success rate will be increased and the hospitalization period will be shortened. In addition to hematopoietic diseases in chemotherapeutic and radioisotope therapies for tumors, thrombocytopenia can also be treated with thrombopoietin.

Considering the various difficulties and known factors described above, the present inventors conducted various studies to find effective factors for the treatment of thrombocytopenia patients or patients with insufficient platelet formation by stimulating the production of platelets, thereby stimulating the differentiation of megakaryocytes. A novel factor was found, a gene encoding these factors was cloned to construct an expression vector, and the gene was expressed to prepare the factor.

Thus, the present invention is directed to (1) stimulating differentiation of megakaryocytes; (2) molecular weight of 55-57 KD and no intermolecular disulfide bond as measured by gel filtration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE); (3) a megakaryocyte differentiation factor having an isoelectric point of 6.5 ± 0.5 and (4) having at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 9 in the sequence list.

The invention also provides a gene encoding a megakaryocyte differentiation factor.

In addition, the present invention provides an expression vector comprising a gene encoding a megakaryocyte differentiation factor.

Furthermore, the present invention provides a host transformed with an expression vector.

The present invention also provides a method for preparing megakaryocyte differentiation factor using the host.

As starting material for separating the megakaryocyte differentiation factor of the present invention, human cancer cells, preferably human epidermal cancer cell A431, particularly preferably human epidermal cancer A431 grown in protein-free medium may be mentioned.

In addition to the above-defined megakaryocyte differentiation factors, the present invention is obtainable from a transformant, such as a cell or animal constructed by genetic technology, wherein the amino acid sequence identical to the above-defined megakaryocyte differentiation factor, and a part of the above-defined megakaryocyte differentiation factor are deleted. Amino acid sequence, a portion of the above-defined megakaryocyte differentiation factor replaced by another amino acid or another amino acid sequence, or one or more amino acid sequences having an amino acid sequence added to the above-defined megakaryocyte differentiation factor, or a combination of the above modifications It relates to a megakaryocyte differentiation factor having an amino acid sequence comprising a.

Furthermore, the present invention provides a megakaryocyte differentiation factor having the amino acid sequence shown in SEQ ID NO: 30, an amino acid sequence in which a portion of the amino acid sequence shown in SEQ ID NO: 30 is deleted, and an amino acid sequence in which a portion of the amino acid sequence shown in SEQ ID NO: 30 is replaced with another amino acid or amino acid sequence. Or to a megakaryocyte differentiation factor having an amino acid sequence in which one or more amino acid sequences have been added to the amino acid sequence shown in SEQ ID NO: 30, or having an amino acid sequence comprising a combination of the above modifications.

The present invention also relates to a gene encoding the aforementioned megakaryocyte differentiation factor. The present invention further relates to a method for producing a megakaryocyte differentiation factor using a gene by genetic recombination techniques. Genetic recombination techniques include the amino acid sequence of a natural megakaryocyte differentiation factor, an amino acid sequence from which a portion of a natural amino acid sequence is deleted, an amino acid sequence in which a portion of a natural amino acid sequence is replaced with another amino acid sequence, or one or more amino acids in a natural amino acid sequence. Performed according to conventional methods using, but not limited to, synthetic or natural polynucleotides encoding amino acid sequences comprising amino acid sequences added or combinations of such modifications.

The various modifications described above can be carried out by conventional techniques such as site-specific mutagenesis.

The number of amino acids included in modifications such as addition, deletion or replacement is not limited, but the amino acid number in addition is, for example, the megakaryocyte differentiation factor of the present invention and the amino acid number of the functional peptide used in the hybrid protein or the factor of the present invention. It depends on the number of amino acids of the signal peptide added to it, ie it depends on the purpose to be modified and in the case of deletion, the number of amino acids can be designed or determined to maintain megakaryocyte differentiation activity, for example, 1 to 30, Preferably 1 to 20, or the number of amino acids in a region other than the active region of the factor of the invention, in the case of replacement the number of amino acids may also be designed or determined to maintain megakaryocyte differentiation activity, for example, For example, it may be 1 to 10, preferably 1 to 5.

Addition or improvement of signal sequences, selection of host-vector systems, and improvement of expression control regions allow for efficient expression. Moreover, the host can be selected to provide glycosylated products. In addition, polynucleotides encoding one or more of the amino acid sequences shown in SEQ ID NOs: 1-9 can be used as DNA probes for cloning genes.

The present invention further provides a pharmaceutical composition comprising a megakaryocyte differentiation factor as an active ingredient. The pharmaceutical composition is preferably used as a medicament for thrombocytopenia.

In addition, the megakaryocyte differentiation factor of the present invention can be used to obtain specific antibodies according to known methods.

Now, the present invention will be described in more detail.

(1) Starting material

Starting materials for obtaining the novel proteins of the present invention include those capable of growing in protein-free medium according to the method of Yamaguchi et al. (See Yamaguchi N. et al., Cancer Res. 50, 7008, 1991). Culture supernatants of cells derived from epidermal cancer cell A431 (ATCC CRL 1555) can be mentioned. This cell line has been designated as human epidermal cancer SBM 330 and is dated July 1, 1992 by Fermeiltation Research Institute Agency of Industrial Science and Technology; Location: 1-3 (307, Higashi 1-chome, Tsukuba City, Ibarakiken), was deposited as FERM BP-3911.

(2) Test method of megakaryocyte differentiation factor

To assay for megakaryocyte differentiation factor, megakaryocyte-based cell lines (eg, CMK follicles or cells derived therefrom), or mouse bone marrow cells, can be used. For example, the activity of acetylcholine esterase known to be specifically detected in murine megakaryocytes can be determined by Ishibashi et al. (See Ishibashi, T. et al, Proc. Natl. Acad. Sci. USA 86, 5953, 1989) using mouse bone marrow cells. Furthermore, harmonious detection of megakaryocytes is performed by acetylcholine esterase staining and May-Göbelt-test staining of cultured bone marrow cells and observing the shape of the stained cells.

(3) purification of megakaryocyte differentiation factor

The megakaryocyte differentiation factor is, for example, starting from the culture supernatant of A431 cells cultured in protein-free medium and concentrated by ultrafiltration, for example, Matrex Blue A (Amicon Co.), Q-Sepharose (Pharmacia), phenyl-sepharose (Pharmacia), S-sepharose (Pharmacia) and Hillroad 26/60 Superdex 75 (Pharmacia) can be purified by column chromatography using alone or in combination. .

Protein is examined by measuring at A280nm.

(4) Determination of partial amino acid sequence of megakaryocyte differentiation factor

To determine the amino acid sequence, the megakaryocyte differentiation factor purified in section (3) is digested into fragments using proteases such as Achromobacter protease I (API) at 37 ° C. for 2 hours. The resulting peptide fragments are separated and recovered by reverse phase HPLC (acetonitrile concentration gradient in 0.1% trifluoroacetic acid) using a YMC-pack AM-303 column. The peptide fragments thus obtained are subjected to an analyzer such as, for example, a gel-phase sequencer obtained from Applied Biosystem. The exact purification method and detailed properties of the megakaryocyte differentiation factor are described in Example 1.

The invention also provides a gene encoding a megakaryocyte differentiation factor. The gene can be cDNA prepared from mRNA, genomic DNA, and synthetic DHA. For example, cDNA is a polymerase using DNA (nucleotide) primers designed based on the partial amino acid sequence shown in Example 1 of human epidermal cancer cells, ie, megakaryocyte differentiation factor purified from the aforementioned human cells, such as A431 cells. Can be cloned by chain reaction (PCR). This cloning method is described in detail in Examples 1 and 2.

In addition, the gene of the present invention includes DNA encoding a protein or glycoprotein having megakaryocyte differentiation activity and hybridizing with the nucleotide sequence of SEQ ID NO: 30. The amino acid sequence expected from the nucleotide sequence and the nucleotide sequence of the cloned DNA in Example 2 is shown in SEQ ID NO: 30.

After determining the amino acid sequence in this way, a polypeptide, one or more amino acids of which variously mutated megakaryocyte differentiation factors, e.g., one or more amino acids, retain the megakaryocyte differentiation factor activity added to the natural amino acid sequence or the amino acid sequence shown in SEQ ID NO: 30 A polypeptide deleted from this amino acid sequence or the amino acid sequence shown in SEQ ID NO: 30 and retaining megakaryocyte differentiation factor activity, at least one amino acid is substituted with one or more other amino acids in the natural amino acid sequence or amino acid sequence shown in SEQ ID NO: 30, Colipeptides that retain megakaryocyte differentiation activity or polypeptides that retain megakaryocyte differentiation factor activity can be designed and produced, including combinations of the foregoing modifications such as addition, deletion and / or substitution of amino acids.

According to the present invention, even if the nucleotide sequence shown in SEQ ID NO: 30 is described, the gene encoding the megakaryocyte differentiation factor of the present invention is not limited thereto. Once the amino acid sequence of the natural megakaryocyte differentiation factor of the present invention or the amino acid sequence of the mutated megakaryocyte differentiation factor is measured, the degeneracy of the genetic code allows the design and preparation of various nucleotide sequences encoding the same amino acid. In this case, codons with a high frequency in the selected host are preferably used.

The gene encoding the megakaryocyte differentiation factor of the present invention can be obtained as cDNA according to Example 2, but this gene is not limited to cDNA. That is, once the nucleotide sequence encoding the amino acid sequence of a native megakaryote differentiation factor is determined, the gene encoding the native megakaryote differentiation factor can be cloned as a cDNA according to a strategy different from the strategy exactly described herein, and moreover. Preferred genes can be cloned from the genome of the cell producing megakaryocyte differentiation factor.

If the gene of interest has been cloned from the genome, the various primer nucleotides or probe nucleotides used in Example 2 can be used as probes for selecting genomic DNA fragments. Moreover, other probes designed based on the nucleotide sequence set forth in SEQ ID NO: 30 can also be used. General methods for cloning the desired DNA from the genome are known in the art (Current Protocols In Molecular Biology, John Wiley & Sons, Chapters 5 and 6).

The gene encoding the natural megakaryocyte differentiation factor of the present invention can be produced by chemical synthesis. Chemically synthesizing DNA using an automated DNA synthesizer, such as the Applied Biosystems 396 DNA / RNA synthesizer, is easy in the art. Thus, one skilled in the art can easily synthesize DNA having the nucleotide sequence shown in SEQ ID NO: 30.

The gene encoding the natural megakaryocyte differentiation factor of the present invention using a codon different from the natural codon, and the gene encoding the mutated megakaryocyte differentiation factor can be prepared by chemical synthesis as described above. Alternatively, these can be obtained by site-specific mutagenesis using DNA or RNA having the nucleotide sequence shown in SEQ ID NO: 30 as a template with mutagenic primers (see Current Protocols In Molecular Biology, John Wiley & Sons). , Chapter 8).

Once the gene encoding the megakaryocyte differentiation factor of the present invention is obtained, the non-gene can be used to prepare recombinant megakaryocyte differentiation factor according to conventional genetic recombination techniques. That is, the DNA encoding the megakaryotic differentiation factor of the present invention is inserted into an appropriate expression vector, the vector is introduced into an appropriate host cell, and then the transformed host cell is cultured and the desired megakaryocyte differentiation factor is cultured (cell or Medium). The megakaryocyte differentiation factor of the present invention may be biochemically or chemically modified, ie, N-terminal acylated.

Furthermore, based on the nucleotide sequence shown in SEQ ID NO: 30, the protein database is searched with a fasta program (GCG package). As a result, it can be seen that the megakaryocyte differentiation factor belongs to the super family of serine protease inhibitors. On the other hand, human leukocyte elastase inhibitors, chicken ovalbumin Y gene product, human plasminogen activator inhibitor 2 and human squamous, similar to the megakaryocyte differentiation factor of the present invention in the expected conformation and distribution of hydrophobic and hydrophilic amino acids. For cell cancer antigens, the N-terminal site is not cleaved and forms a signal peptide. Thus, in the megakaryocyte differentiation factor of the present invention, the N-terminal site can act as a signal peptide and the megakaryocyte differentiation factor can be secreted without cleavage of the signal peptide. The megakaryocyte differentiation factor of the invention can also be modified such that the first methionine is deleted and the second alanine is acetylated.

As the host, both prokaryotic and eukaryotic cells can be used. As prokaryotic cells, bacteria such as Bacillus genus such as Escherichia coli and Bacillus Subtilis can be used. As eukaryotic cells, yeasts such as Saccharomyces spp. Seccharomyces cerevisiae], Spodoptera frugiperda cells, Cabbage looper cells, insect cells such as Bombyx mori cells, human cells Animal cells such as monkey cells, mouse cells and the like can be used. Furthermore, insect cells such as Bombyx mori can be used.

As the expression vector, plasmids, phages, phagemids, baculoviruses, viruses such as Basinia virus and the like can be used. The promoter in the expression vector is selected according to the host used. For example, lac promoter, trp promoter and the like can be used as the bacterial promoter, adhl promoter, pqk promoter and the like can be used as the yeast promoter. On the other hand, the baculo virus polyhedrin promoter can be used as an insect promoter and the Simian virus 40 early or late promoter can be used as a promoter for animal cells.

Transformation of a host using an expression vector can be performed according to conventional methods known in the art, which methods are described in Current Protocols in Molecular Biology, John Wiley & Sons. Cultivation of the transformants is also carried out according to conventional methods.

Purification of megakaryocyte differentiation factor from culture or insect bodies is carried out using conventional methods used for isolation and purification of proteins, for example, ultrafiltration, column chromatography in various forms such as Q-sepharose column chromatography, and the like. Can be done.

Example

The present invention will now be described in more detail by way of examples.

Example 1

Purification of Megakaryocyte Differentiation Factor

(1) Culture of A431 Cells

Frozen SBM 330 cells conditioned in protein-free medium from A431 cells were thawed and cultured in primary medium (Ham's F12 medium containing 10% calf serum). That is, the cells are plated in a 10T polar flask having a culture area of 150 cm ² and cultured so as to be colonized at 37 ° C. in the presence of 5% CO ₂ . Next, the cells are scattered with 0.25% trypsin solution (Chiba Kessei; manufacturer) and subcultured at 37 ° C. and 0.5 rpm for about 3 days in 10 roller bottles having a culture area of 850 cm ² to recover 1 8 × 19 ⁹ cells. . The cultured cells are attached to a ceramic core (S-451) of an Opti-cell incubator (Wilmington, Mass., Charles River Inc.) and perfusion culture using 10 L of primary medium.

Perfusion cultures are performed at 37 ° with oxygen at 150 mmHg. The primary medium is then replaced with protein-free medium. After 7 days of incubation in primary medium, the protein-free medium is fed to the culture at a rate of 20 L / day and at the same time the culture supernatant is recovered from the culture at the same rate. As a result, the serum-containing primary medium is substantially completely replaced with the protein-free medium by feeding about 100 L of protein-free medium. Thereafter, the cell culture supernatant is continuously collected to obtain 1000 L of the cell culture supernatant. A portion of the cell culture supernatant thus obtained (about 300 L) was concentrated to 2 L using an ultrafiltration membrane (see Milipore, Bedford, MA; 10,000 cuts in MW) and the concentrate was concentrated to 20 mM Tris / HCl buffer (pH 7.4). Dialysis

(2) Assay of megakaryocyte differentiation factor using mouse bone marrow cells

Bone marrow cells are extracted from the thighs of BDF _I , female mice and suspended in α-MEM medium (McLean, VA, USA, Flow Laboratories, Inc.). Percoll layers of different densities (Pharmacia LKB Biotechnology, Tokyo, Japan) are applied, and bone marrow cell suspensions are placed there and centrifuged at 400 g for 20 minutes. Collect mononuclear cells from the surface of the layer with a density of 1.07 g / ml, collect the layer with a density of 1.08 g / ml, wash once with α-MEM containing 10% FBS, and 0.5 mM diisopropyl fluorophosphate. Resuspend in the same medium containing. The suspension is then placed in a plastic cell culture dish (Corning, NY, USA) and incubated for 2 hours at 37 ° C. in 5% CO 2 and 95% air. During the incubation, one hour after incubation, the cell culture dish is replaced with a new one. After incubation, cells are washed three times with 10% FBS / α-MEM.

The non-adherent myeloid mononuclear cells thus obtained were suspended in 10% FBS / 1% BSA / 0.1 mM 2-mercaptoethanol / α-MEM and 5 × 10 ⁴ cells / in 96-well microplates (Corning). Smear in the amount of well. If necessary, test samples were treated with 25 U / ml mouse recombinant IL-3 (Genzyme Corporatcon, Cambridge, MA, USA) and 1-2 μg / ml anti-IL-6 antibody (Boehringer Mannheim, Mannheim, FRG). Add. If an anti-IL-6 antibody is added, the test sample and antibody are preincubated at 37 ° C. for 1 hour before inoculating the cells.

Incubation is carried out for 4-5 days at 37 ° in 5% CO2-5% O2-90% N2. After culturing the cells in each well of the microplates, they were washed twice with PBS, 0.2% (w / v) Triton X-100 180μ1, 1mM EDTA, 0.12M NaCl, 50mM HEPES (ph 7.5), and substrate Add 20μ 1, 5.6mM acetithiolchlor iodide. After incubation for 1 hour with shaking at room temperature, 20 μl of this solution is transferred to microplates for fluorescence analysis (Dynatech Micro FLUOR "B" plate).

0.4 micron 7-diethylamino-3- (4'-maleimidylphenyl) -4-methylcoumarin 20μ 1, 0.2% (w / v) Triton X-100 160μ 1, 1mM EDTA in acetonitrile , 50 mM sodium acetic acid (PH 5.0) was added and fluorescence emission was measured with a fluorometer (excitation 365 nm, emission 450 nm).

For acetylcholine esterase staining, the cells are centrifuged with Cytospin to attach the cells on a slide glass, fixed with 5% glutaraldehyde, 10 mM phosphate buffer (pH 6.7) for 15 minutes, and the substrate Acetylthiocholine is used according to the Mizoguchi method (Method for Culturing Hemopoietic Stem Cells, Chugai Igaku, 1986, ed. Y. Miura, pp 82-88). That is, after the cells were fixed, the slide glass was washed with 0.1 M phosphate buffer (pH 6.0), and each slide glass was 0.67 mg / ml acetylthiocholine iodide 1.5 ml, 0.1 M phosphate buffer (pH 6.0), 30 mM 0.2 ml of CUSO4, 0.2 ml of 5 mM potassium ferrocyanide and 0.1 ml of 0.1 M sodium citrate are stacked. The slide glass is incubated at room temperature for 4 hours and washed with water. The May-Grouenwald-Gimesa staining method is well known in hematology. It is performed using reagents commercially available from Merck (E, Merck, Darmstaclt, FRC), with May-Grouen Wald staining for 4 minutes and Gimsa staining for 10 minutes.

(3) purification of megakaryocyte differentiation factor

The supernatant of the cultured A431 cells was dialyzed and centrifuged to obtain supernatant, which was applied to a Matrex Bluc A Column equilibrated with 20 mM Tris / HCl buffer (pH 7.4) and the column was buffered with the same buffer. After washing, the bound fractions are eluted with the same buffer containing 2M NaCl. The megakaryocyte differentiation activity detected by the method described above appears in the binding fraction. Thus, the binding fraction is applied to a Q-Sepharose column which is dialyzed with 20 mM Tris / HCl buffer (pH 7.4) and equilibrated with the same buffer. The column is washed thoroughly and the megakaryocyte differentiation factor is eluted with a NaCl gradient (see FIG. 1). This factor elutes near about 0.3-0.5 M NaCl concentration.

The active fraction obtained from Q-Sepharose is made into 30% saturated ammonium sulfate and applied to a phenyl Sepharose column equilibrated with 20 mM Tris / HCl buffer (pH 7.4) containing 30% saturated ammonium sulfate. The megakaryocyte differentiation factor is coagulated by simultaneously forming a concentration gradient of ammonium sulfate (30% to 0%) and ethylene glycol (0 to 50%) (FIG. 2). The megakaryocyte differentiation factor measured in the presence of anti-IL-6 antibody is observed on several fractions early in the gradient formation.

The fractions thus obtained are mixed and completely dialyzed with 50 mM MES / NaOH buffer (pH 6.0) and applied to an S-Sepharose column equilibrated with the same buffer. The binding fraction is eluted by a 0 to 0.5 M NaCl gradient (see FIG. 3). The activity is widely distributed, but relatively high activity appears at the beginning of the concentration gradient formation. Fractions obtained from S-Sepharose are applied to Hill Road 26/60 Superdex 75 (Pharmacia) for gel filtration. The column used is previously equilibrated with the same buffer, eluted with the same buffer (see Figure 4), and the eukaryotic differentiation activity is eluted near the position corresponding to a molecular weight of 55 to 57 KDa.

Approximately 80 μg of a fraction showing two bands near 55-57 KDa analyzed by SDS-PAGE is obtained from 300 L of culture supernatant of A431 induced cells according to the steps described above (FIG. 5). Two bands are associated with activity (Figure 1). Thus, it can be concluded that the two bands observed in this fraction are observed in the fraction corresponding to the desired megakaryocyte differentiation factor.

(4) Characteristics of megakaryocyte differentiation factor

This megakaryocyte differentiation factor has the following characteristics.

1) Molecular weight: about 55KDa (gel filtration and SDS-PAGE) (FIGS. 4 and 5)

This factor shows two bands in SDS-PAGE and there is no difference in migration under reducing or non-reducing conditions. Therefore, this factor does not have intermolecular disulfide bonds.

2) Isoelectric point: 6.5 ± 0.5 (figure 6)

Several bands are observed in the above-described range.

3) The heterogeneity of the present factor can be explained as heterogeneity in the sugar chain structure of the glycoprotein. In other words, in the case of the factor treated with the asparagine bound sugar removing enzyme endoglycosidase F, the molecular weight of this factor is reduced to about 40 KDa in molecular weight, and also heterogeneity is reduced in SDS-PAGE (Fig. 7). In addition, fractions representing single bands and fractions representing two bands were digested with API and peptide maps were prepared by fractionation by reverse phase HPLC, and no difference was observed between the two fractions.

4) This factor contains one or more amino acid sequences shown in SEQ ID NOs: 1-9.

5) biological activity

When mouse bone marrow cells are cultured in the presence of purified megakaryocyte differentiation factor and IL-3, the proliferation and differentiation of megakaryocytes is observed (FIGS. 8, 9 and 10). 8 shows measurement results for acetylcholine esterase activity of megakaryocytes; 9 shows the results of acetylcholine esterase staining of cultured cells (X20); 10 shows the results of May-Grouenwald-Gymsa staining of cultured cells (X100). In both Figures 9 and 10, megakaryocytes show an increase in their presence (B) compared to the absence of a megakaryocyte differentiation factor (A).

(5) structure of megakaryocyte differentiation factor

To characterize the structure of the purified megakaryocyte differentiation factor, this factor is digested with API to determine the structure of the resulting fragment. After factor degradation with API, each fragment is recovered by reverse phase HPLC and the structure for the appropriate fraction is determined. As a result, the peptide fragment has the amino acid sequence shown in SEQ ID NOs: 1-9.

Example 2

Determination of cDNA Structure for Megakaryocyte Differentiation Factor

1. Analysis of cDNA nucleotide sequence of megakaryocyte differentiation factor by PCR (1)

Oligonucleotides NI 065 (SEQ ID NO: 10; corresponding to 449 through 486 of SEQ ID NO: 30) and NI 067 (SEQ ID NO: 11; corresponding to 1049 through 1080 of SEQ ID NO: 30) based on the amino acid sequence shown in SEQ ID NOS: 3 and 4, respectively, It is synthesize | combined by devising.

Total RNA is purified from A431 cells using Wako Pure Checmical (ISOGEN) according to the manufacturer's instructions. RNA with poly A is purified from total RNA and the reaction is performed using the 3'-RACE Kit (Gibco BRL). That is, using the oligomer 3'-RACE adapter primer (SEQ ID NO: 12) attached to the oligomer NI 065 and 3'-RACE Kit (Gibco BRL) described above, polymerase chain reaction (PCR) was performed according to the instructions included with the kit. Perform.

The reaction product is then pra and secondary PCR using oligomeric 3'-RACE adapter primers included in NI067 and 3'-RACE Kit (Gibco BRL) to obtain DNA fragments with about 900 base pairs. Then, using nucleotide sequencing directly on the PCR product, U. Gyllensten et. al., Proc. Natl. Acad. Sci USA 85: 7652 (1988)], using a DNA fragment with about 900 base pairs as a direct reaction substrate, a Taq Dye Deoxy Terminator kit (Taq Dye Deoxy Terminator) commercially available from Applied Biosystems according to the manufacturer's instructions. Cycle Seguencing Kit) and fluorescent nucleotide sequencer (Applied Biosystem, Type 370 A) are used to determine the nucleotide sequence of the portion representing the protein and the downstream portion of the protein portion. As a result, the sequences from nucleotides 1081 to 1950 of SEQ ID NO: 30 are shown.

Based on this sequence, oligomer KY100 (SEQ ID NO: 13 corresponds to 1255 to 1236 of SEQ ID NO: 30) is synthesized. Further PCR using NI 065 and KY 100 is performed using the reaction product obtained by PCR as oligomeric 3'-RACE adapter primer attached to the NI065 and 3'-RACE Kit (Gibco BRL) as the reaction substrate. . As a result, a DNA fragment having 807 base pairs is obtained.

DNA fragments having such 807 base pairs are used as direct reaction substrates to determine their nucleotide sequences using the Tac Di Deoxyterminator Cycle Sequencing Kit and Fluorescent Nucleotide Sequencer, commercially available from Applied Biosystems. As a result, the nucleotide sequence from nucleotides 487 to 1080 of SEQ ID NO: 30 is shown. Based on this sequence, oligomeric NI073 (SEQ ID NO: 14; corresponds to 864-886 of SEQ ID NO: 30), NI074 (SEQ ID NO: 15; corresponds to 1012-992 of SEQ ID NO: 30), and NI075 (SEQ ID NO: 16; 802 of SEQ ID NO: 30). To 782).

2. Analysis of cDNA nucleotide sequence of megakaryocyte differentiation factor by PCR (2)

A. Preparation of mRNA from megakaryocyte differentiation factor expressing cell line (A431)

25 μg of mRNA is extracted from frozen cells of human epidermal cancer cell line (A431) and purified using RNA extraction kit and mRNA purification kit available from Pharmacia LKB.

B. Preparation of DNA phage library from megakaryocyte differentiation factor expressing cell line (A431)

(1) Synthesis of cDNA

From 5 μg of mRNA derived from A431, cDNA is synthesized using the cDNA Synthesis Kit Time Saver, available from Pharmacia-LKB. 5 μg of mRNA dissolved in 20 μl of distilled water treated with diethylpyrocarbonate (DEPC) is first heated at 65 ° C. for 10 minutes and cooled on ice. To this was added 11μ 1 of the first strand reaction mixture, 1μ 1 of the DTT solution and 1μ 1 of the 130U / ml NotI / oligomer 18 primer solution (Pamasia-LKB), and the mixture was incubated at 37 ° C for 1 hour.

The reaction mixture is added to the second strand reaction mixture, which is incubated at 12 ° C. for 30 minutes, 22 ° C. for 1 hour, and heated at 65 ° C. for 10 minutes. Phenol / chloroform / isoaniyl alcohol (25: 24: 1; then abbreviated PC) 100 μl was added to this, the mixture was vigorously stirred and centrifuged at 14,000 × g for 1 minute to give a supernatant. This was fractionated by Sephacryl S-400 spin column (Pharmacia LKB) to obtain 100 µ1 of cDNA solution.

(2) Addition of EcoRl Adapter

To 100 μ1 of cDNA solution, 5 μl of 10 U / ml EcoRl adapter (Pharmacia, LKB), 30 μl of polyethylene glycol buffer, 1 μ1 of diluted ATP solution and 1 μ1 of T4 DNA ligase and 1 μ1 of T4 DNA ligase were added, and the mixture was subjected to 1 at 37 ° C. Incubate for hours. After heating at 65 ° C. for 10 minutes, 1.5 μl of ATP solution and 1 μl of T4 polynucleotide kinase are added thereto and the mixture is incubated at 37 ° C. for 30 minutes. After heating at 65 ° C. for 10 minutes, 20 U / μ 1 NotI 2 μ 1 is added to this mixture and then incubated at 37 ° C. for 1 hour. 150 μl of PC was added to the mixture, stirred vigorously and centrifuged at 14,000 × g for 1 minute to fractionate this supernatant on a Sephadex S-400 spin column to give 150 μl of cDNA solution.

(3) Incorporation of cDNA into phage vectors and in vitro packaging

After digestion with EcoRI and NotI, 2 μg of dephosphorylated λ gt110 (Pharmacia LKB) was added to 15 μ1 of cDNA solution. After ethanol precipitation, the precipitate was dissolved in 8 μl of ligase buffer, 1 μl of 1/75 diluted ATP solution and 1 μl of T4 DNA ligase were added to the solution, followed by incubation at 16 ° C. for 30 minutes and on ice Save it.

In vitro packaging reactions are performed using GigapackII Gold Stratagene and a recombinant phage 3.22 × 10 6 pfu library is obtained from these three ligase reaction products. This library. Amplification in E. coli Y1090r host yields A431 phage library stock 6.0 × 10 10 pfu / ml.

C. Identification and isolation of cDNA fragments for megakaryocyte differentiation factor by PCR

(1) Amplification of cDNA Insertion Fragments in A431 Phage Library by PCR

¹⁰ μl of stock solution (corresponding to 6.0 x 10 ⁸ pfu) of a 6.0 x 10 ¹⁰ pfu / ml A431 phage library was used as template DNA for PCR reactions, and 10 x PCR buffer 5 μ1, 1.25 mM 4dNTPs 8 μ1, 10 D / ml λ gt11-forward primer (λ gt 11F) (SEQ ID NO: 17) 2μ 1, 10D / ml A gt 11-reverse primer (A gt 11R) (SEQ ID NO: 18) and 5U / μ 1 Taq DNA polymerase (Perkin Elmer Cetus) 1μ 1 is added and the total volume of this mixture is brought to 50μ 1 with DEPC-treated distilled water. Thirty reactions were carried out at 93 ° for 1 minute, at 55 ° C. for 2 minutes and at 72 ° C. for 3 minutes, and the reaction mixture was incubated at 72 ° C. for 10 minutes. The results of the analysis by 1% agarose gel electrophoresis show a speckle pattern ranging from 0.8 to 6 kb.

(2) using cDNA insert DNA amplification fragment mixture as a template and TP7 (SEQ ID NO: 20; corresponding to 683-703 of SEQ ID NO: 30), / TP10, TP7 / TP6 (SEQ ID NO: 19; corresponding to 1036-1001 of SEQ ID NO: 30) as primers PCR analysis using TP8 (SEQ ID NO: 21; corresponding to 941 to 964 of SEQ ID NO: 30), TP10 (SEQ ID NO: 22; corresponding to 1036 to 986 of SEQ ID NO: 30), and TP8 / TP6

1 μl of a 1 / 5000-diluted solution of the PCR reaction product described above was used as template DNA for PCR reactions, 10 μg PCR buffer 5 μl, 1.25 mM 5 dNTPs 8 μl, 2 μl of 10D / ml primers, respectively, in the combinations described below. 1 and Perfect Match (Stratagene) 1 μ 1 are added and the total volume of this mixture is brought to 49 μ 1 with DEPC-treated distilled water.

The reaction mixture is heated at 95 ° C. for 5 minutes and at 60 ° C. for 5 minutes and 5 U / μ 1 Taq DNA polymerase (Perkin Elmer Cetus) 1 μ 1 is added thereto, 1 min at 94 ° C., 2 min at 60 ° C. And reacted 30 times at 72 ° C. for 3 minutes and then incubated at 72 ° C. for 10 minutes. As primers, TP7 / TP10, TP7 / TP6, TP8 / TP10, and TP8 / TP6 are used.

As a result of analysis of the PCR reaction product by 2% agarose gel electrophoresis, 354 bp, 354 bp, 96 bp and 96 bp bands are respectively obtained corresponding to the primers.

(3) Analysis of the primary sequence of the PCR amplification product (354 bp) obtained by using the primers TP7 / TP10 and TP7 / TP6

Bands of PCR amplification products (354bP) obtained by using primers TP7 / TP10 and TP7 / TP6 were electrophoresed, cut out from 2% agarose gel, and 50 μl of DEPC-treated distilled water was added to the cut agarose gel fragments. And the mixture is heated at 45 ° C. for 30 minutes. 2 μ1 of this solution as template DNA was added 10 μl PCR buffer 5 μ1,1.25 mM 4dNTPs 8 μ1, 2 μl of 10D / ml primer and 1 μl Perfect Match (Stratagene) in combination, and the total volume of the reaction mixture was DEPC-treated. Make 49μ1 with distilled water. The mixture is heated at 95 ° C. for 5 minutes, at 60 ° C. for 5 minutes, 5 U / μ 1 Tap DNA polymerase (Perkin Elmer Cetus) 1 μ 1 is added, 1 minute at 94 ° C., 2 minutes and 72 minutes at 60 ° C. The reaction is carried out 30 times for 3 minutes at ℃, then incubated for 10 minutes at 72 ℃. As primers for the above-described PCR reactions, TP7 / TP10 and TP7 / TP6 are used. Bands of PCR reaction products (354 bp each) were cut out from 2% agarose gels in electrophoresis, extracted and purified to insert the product into pCR II (Invitrogen), followed by transfection of this E. coli INVα F '(Invitrogen). Used to switch Plasmid DNA was extracted and purified to confirm that 354 bp DNA fragment was inserted by EcoRI digestion.

The primary sequence of the DNA insertion fragment is analyzed using M13 forward primer (M13F) (SEQ ID NO: 23) and M13 reverse primer (M13R) (SEQ ID NO: 24) (Automated Sequencer of Applied Biosystem, Model 370A). As a result, a 296 bp sequence corresponding to nucleotides 704 to 999 of SEQ ID NO: 30 was found, which sequence was the C-terminal three amino acids of SEQ ID NO: 9 corresponding to the downstream portion of primer TP7 (XRK: but ERK from DNA nucleotide sequence). , N-terminal five amino acids of SEQ ID NO: 6 corresponding to the upstream portion of primer TP6 (ADLSG), and eight amino acids of SEQ ID NO: 5 corresponding to primer TP8 (YLRALGLK), and the PCR reaction products (354bp each) Identify part of the cDNA encoding differentiation factor.

3. cDNA screening encoding megakaryocytic differentiation factor

A. Preparation of cDNA plasmid library from megakaryocyte differentiation factor expressing cell line (A431)

(1) Synthesis of First Strand cDNA

CDNA is synthesized using a Super Script plasmid system commercially available from GIBCO from 5 μg of mRNA derived from the A431 cell line. First 2 μl of NotI primer adapter is added to 5 μg of mRNA dissolved in 5 μl of distilled water treated with diethylpyrocarbonate (DEPC), and the mixture is heated at 70 ° C. for 10 minutes and cooled on ice. 5 x first strand buffer 4μ 1, 0.1M DTT solution 2μ 1, 10mM dNTPs 1μ 1 and 1μ 1 of distilled water treated with DEPC are added thereto and the mixture is incubated at 37 ° C. for 2 minutes. Superscript reverse transcriptase 5μ 1 is added to this reaction mixture, then incubated for 1 hour at 37 ° C and then placed on ice to stop the reaction.

(2) Synthesis of Second Strand cDNA

DEPC-treated distilled water 93μ1, 5 x second strand buffer 30μ1, 10mM 4NTPs 3μ1, 10U / μ1 in 18μ1 of 20μ1 reaction mixture for first strand cDNA synthesis. 1 μl of E. coli DNA ligase, 4 μl of 10 E / μ 1 E. coli DNA polymerase and 1 μl of E. coli RNase H are added, and the mixture is incubated at 16 ° C. for 2 hours. 2μ 1 (10U) of T4 DNA polymerase is added to this reaction mixture and then incubated at 16 ° C for 5 minutes.

Place the reaction mixture on ice, add 0.5M EDTA 10μ 1 and PC 150μ 1 to it, stir vigorously, centrifuge at 14,000 xg for 10 minutes, and transfer supernatant 140μ 1 to a new centrifuge tube. . 70 μl of 7.5 M ammonium acetate and 0.5 ml of ethanol are added to this supernatant and then left at −80 ° C. for 30 minutes. The mixture is centrifuged at 14,000 x g for 10 minutes, the supernatant is removed and the precipitate is washed with 0.5 ml of 70% ethanol and dried under reduced pressure.

(3) Addition of BstXI Adapter

The cDNA precipitate described above was dissolved in 25μ 1 of DEPC treated distilled water, 10μ 1 of 5x T4 DNA ligase buffer, 10μ 1 of BstXI adapter (Invitrogen) and 5μ 1 of T4 DNA ligase were added to this solution and then at 16 ° C. Incubate for 16 hours. PC 50μ 1 is added to this mixture and stirred vigorously to transfer to a centrifuge tube at 14,000 × g for 5 minutes. 25 μl of 7.5 M ammonium acetate and 150 μl of ethanol are added to this tube and allowed to stir for 30 minutes at −80 ° C. After removing the supernatant by centrifugation at 14,000 x g for 10 minutes, the precipitate is washed with 0.5 ml of 70% ethanol and dried under reduced pressure.

(4) NotI decomposition

The cDNA precipitate described above is dissolved in 41 μl of DEPC treated distilled water, 5 ml of REAct 7 buffer and 4 μl of Notl 4 are added to this solution and incubated at 37 ° C. for 2 hours. PC 50μ 1 is added to this mixture, stirred vigorously and centrifuged at 14,000 × g for 10 minutes, and the supernatant 45μ 1 is transferred to a centrifuge tube.

(5) Adapter removal and partial cDNA size fractionation

The cDNA solution described above is fractionated using Qulck Spin column linker 5 (Boehringer Mannheim). Obtain 40 μg / μ 1 cDNA 50 μ 1.

(6) introduction of cDNA into phage vector and E. E. coli transformation

To 37.5 μ1 of cDNA solution described above, 12.5 μ1 of pCR / CMV (Invitrogen) vector (29 μg / μ1) digested with NotI and BstXI was added, and further 400 μl of Takakara Ligation Kit A solution and 50 μl of B solution is added and the mixture is incubated for 30 minutes at 16 ° C. and transformed into 1 ml of Max Efficiency DH5α competent cells (BRL) to yield 71,550 recombinant clones. All colonies are collected from the plates (2,86 × 10 7 cells / ml) and stored at −80 ° C. in the presence of 20% glycerol.

B. Screening of megakaryocyte differentiation factor cDNA by colony hybridization

Using a cDNA plasmid library derived from the A431 cell line, a total of 227,000 (3700 / plate) colonies were formed on a 9 cm diameter 60 plate and replicated with a nitrocellulose filter. PCR (as described above) using primers NI 067 and 3'-RACE adapter (GIBCOBRL) yielded a 900 bp PCR product, and the PCR product was digested with BamHI to yield two DNA fragments (0.5 kb and 0.4 kb). Probes are prepared by nick-decoding DNA fragments with [α- ³² p] dCTP.

For colony hybridization, the filters were placed in 5 × SCC, 25 mM phosphate buffer (pH 7.4), 5 × Denhaldt solution, 1% SDS, 100 μg / ml of heat denatured salmon sperm DNA and 50% formamide. Incubate for 18 hours at ° C and wash 20 min at 40 ° C. and 20 min at 45 ° C. with 5 × SSC, 0.1 SDS. Detection is by exposing BAS 2000 (Fuji Film) for 18 hours.

First, second and third screening is performed to shrink four clones, eg, TF290, TP308, TP310 and TP317. The length of the inserted cDNA is 1,2 kb, 1.1 kb, 1,2 kb and 1.2 kb, respectively. TF290, TP310 and TP317 encompass the region downstream from nucleotide 685 of SEQ ID NO: 30.

4. Analysis of cDNA nucleotide sequences encoding megakaryocyte differentiation factor by FCR (3)

A. Preparation of mRNA from megakaryocyte differentiation factor expressing cell line (HPC-Y11)

From 1.1 g of frozen cells of the human pancreatic cancer cell line (HPC-Y11), 50 μg of mRNA is extracted and purified using an RNA extraction kit and an mRNA purification kit available from Pharmacia LKB.

B, Preparation of cDNA Phage Library from Megakaryocyte Differentiation Factor Expressing Cell Line (HPC-Y11) (1) Synthesis of cDNA

From 5 μg of mRNA derived from HPC-Y11, cDNA is synthesized using Pharmacia LKB's time saver cDNA synthesis kit. First, 5 μg of mRNA is dissolved in 20 μl of diethylpyrocarbonate (DEPC) treated distilled water and the solution is heated at 65 ° C. for 10 minutes and cooled on ice. 11 μl of the first strand reaction mixture, 1 μl of DTT solution and 1 μl of NotI (oligomer 18 primer solution) are added to the mixture and then incubated at 37 ° C. for 1 hour.

100 μl of the second strand reaction mixture is added to this mixture, followed by incubation at 12 ° C. for 30 minutes and 22 ° C. for 1 hour, and heating at 65 ° C. for 10 minutes, phenol-chloroform-isoamyl alcohol (25 : 24: 1, then abbreviated as PC) 100 μ 1 was added to this mixture, stirred vigorously and centrifuged at 14,000 × g for 1 minute, and the supernatant was washed with a Sephacryl S-400 spin column (Pharmacia-LKB). Fractionation to give 100 μl of cDNA solution.

(2) Addition of EcoRI Adapter

To 100 μl of cDNA solution is added 5 μl of EcoRI Adapter (Pharmacia-LKB), 30 μl of polyethylene glycol, 1 μl of ATP solution and 1 μl of T4 DNA ligase, and the mixture is incubated at 37 ° C. for 1 hour. After 10 minutes of heating at 65 ° C., 1.5 μl of ATP solution and 1 μl of T4 polynucleotide kinase are added to this mixture and incubated at 37 ° C. for 30 minutes. Heat at 65 ° C. for 10 minutes, add NotI 12μ 1 to this mixture, and incubate at 37 ° C. for 1 hour. 150 μl of PC was added to this mixture, stirred vigorously, centrifuged at 14,000 × g for 1 minute and the supernatant fractionated using a Sephacryl S-400 spin column to give 150 μl of cDNA solution.

(3) Introduction of cDNA as a phage vector and in vitro packaging

2 μg of λ gt 11D (Pharmacia-LKB) which was dephosphorylated by EcoRI and NotI and dephosphorylated was added to 15 μ1 of the cDNA solution, followed by ethanol precipitation, and the precipitate was dissolved in 8 μ1 of ligase buffer. 1 μl of 1 / 75-diluted ATP solution and 1 μl of T4 PDNA ligase are added to this mixture, then incubated at 16 ° C. for 30 minutes and stored on ice. In vitro packaging reactions are carried out using Giga Pack II Gold (Stratagene) and recombinant phage 5.34 × 10 ⁶ pfu is obtained from the three ligase reaction products described above. The library is amplified in this Coli Y 1090r-1 host to obtain a stock of 1.7 × 10 11 pfu / ml HPC-Y11 phage library.

C. Identification and Isolation of the 5'-Part of the Megakaryocyte Differentiation Factor cDNA

(1) Amplification of HPC-Y11 Phage Library cDNA Insertion DNA Fragment Primer N1074 Upstream by PCR

As template DNA for PCR reactions, 1 μl of 6.0 x 10 ¹⁰ pfu / ml HPC-Y11 phage library stock solution (corresponding to 1.7 x 10 ⁹ pfu) 5 μl of 10 × PCR buffer, 1 μl of 1.25 mM 4 dNTPs, 10 0D / 1 μl of ml λ gt 11-forward F1 primer (SEQ ID NO: 25), 1 μ1 of 50 D / ml N1074 primer, and 1 μ1 of Perfect Matat (Stratagene) are added and the total volume is made 49 μ1 of distilled water treated with DEPC.

The reaction mixture was heated at 95 ° C. for 5 minutes and at 60 ° C., then 5 U / μ 1 Taq DNA polymerase (Perkin Elmer Cetus) 1 μ 1 was added thereto, and at 94 ° C. for 1 minute, 60 React 35 times at 1 ° C. for 10 minutes at 72 ° C. The analysis by 2% agarose gel electrophoresis showed a stained pattern in the range of 0.3 to 6 kb.

(2) a template which is a mixture of PCR amplification fragments prepared using the λ gt11F1 / NI074 primers, and λ gt11F2 (SEQ ID NO: 26) / NI075, λ gt11F2 / TP12 (SEQ ID NO: 28; corresponding to 703-683 of SEQ ID NO: 30), as a primer PCR using λ gt11F2 / TP11 (SEQ ID NO: 27; corresponding to 619-599 of SEQ ID NO: 30), λgt11F2 / TP13 (SEQ ID NO: 29; corresponding to 595-575 of SEQ ID NO: 30), TP7 / NI074, TP7 / NI075, and NI073 / NI074 analysis

1 μl of the l / 100 diluted solution of the PCR reaction product is used for template DNA for PCR reactions, 5 μl 10 × PCR buffer, 8 μl of 1.25 mM 4dNTP, each 10 0D / ml primer mixed as described below 0.5 μl and 1 μl Perfect Match (Stratagene) are added thereto and then the entire reaction mixture is brought to 49 μl using DEPC-treated distilled water. The reaction mixture is heated at 95 ° C. for 5 minutes and at 60 ° C. for 5 minutes and 1 μl of 5 U / μl Taq DNA polymerase (Perkin Elmer Cetus) is added thereto, followed by 1 minute at 94 ° C., 2 minutes at 60 ° C. and 72 ° C. The reaction was performed 35 times for 2 minutes at and then incubated at 72 ° C for 10 minutes.

Lambda gt11F2 / NI075, lambda gt11F2 / TP12, lambda gt11F2 / TP11, lambda gt11F2 / TP13, TP7 / NI075, and NI073 / NI074 as primers. 2% agarose gel electrophoresis results in bands of 969 bp, 870 bp, 786 bp, 762 bp, 330 bp and 149 bp corresponding to the primers.

(3) PCR analysis and primary sequence analysis using λ gt11F / TP11 and λ gt11F / TP13 primers for PCR amplification products (969 bp) prepared using F2 / NI075 primers

0.5 μl of the PCR reaction product (969bP) prepared using λ gt11F2 / NI075 as a primer was used as template DNA for the PCR reaction, and 5 μl of 10 × PCR buffer, 8 μl of 1.25 mM 4dNTP, 10 0D / ml λ gt11F1 primer 1 1 μl, 1 μl of 10 0D / ml TP11 primer or 1 μl of 10 0D / ml TP13 primer and 1 μl of Perfect Match are added thereto, followed by 49 μl total volume using DEPC-treated distilled water.

The reaction mixture was heated at 95 ° C. for 5 minutes and at 60 ° C. for 5 minutes, and 1 μl of 5 U / ml Taq / DNA polymerase (Perkin Elmer Cetus) was added thereto, followed by 1 minute at 94 ° C., 2 minutes at 72 ° C. and 72 minutes. The reaction is carried out 35 times at 2 ° C. for 10 minutes at 72 ° C.

Bands of the PCR reaction product (678 bp and 654 bp, respectively) were cut from 2% agarose gel after electrophoresis, extracted, purified, inserted into pCRII (Invitrogen), and this coli IN Vα F '(INtrogen) Used to transform. Plasmid DNA is extracted, purified from the transformants, and digested with EcoRI to confirm that a 0.75 kb DNA fragment is inserted. The primary sequence of the inserted DNA fragment is analyzed using M13 forward primer M13F and M13 reverse primer M13 (Applied Biosystems automated sequencer Model 370A).

As a result, 619 bp of sequences corresponding to nucleotides 1 to 619 of SEQ ID NO: 30 appear, and the nucleotide sequence of 133 nucleotides consisting of nucleotides 487 to 619 of SEQ ID NO: 30 is located at the N-terminus of the primary sequence shown in Example 2.1. Corresponds. Among these 619 bp sequences are 19 amino acids of SEQ ID NO: 3 (VERVDFTNHLEDTRRINK from DNA nucleotide sequence) and 5 amino acids of SEQ ID NO: 7 (LYDAK), and these PCR reaction products (0.7 kb each) represent the cDNA encoding megakaryocyte differentiation factor. It is obvious that it is part.

Translation initiation methionine corresponds to nucleotide 74 and the 5'-non translation region is considered to consist of 73 bp. Thus, it is evident that these PCR reaction products (0.7 kb each) contain the N-terminus of the structural gene for the megakaryocyte differentiation factor.

5. cDNA nucleotide sequence analysis encoding megakaryocyte differentiation factor by PCR (4)

Among the sequences considered to be the 5'-non-translated region obtained in section C (3) and the N-terminal region of the structural gene for the megakaryocyte differentiation factor derived from HPC-Y11, the 5'-non-detox region The nucleotide sequences of Nos. 12 to 31 of SEQ ID NO: 30 are used as the basis for oligomer Ni 083 (SEQ ID NO: 31) synthesis.

Synthesizing the first strand cDNA according to the instructions included using RNA containing poly A prepared from A431 cells in section 1, a preamplification system (Gibco BRL) and a random hexamer attached to the system, NI 083 and NI PCR is performed using 074 and Ampli Tas (Takara). As a result, a DNA fragment of 1001 bp, which is a cDNA fragment for megakaryocyte differentiation factor, is obtained.

DNA fragments of this PCR product, 1001bP, are used directly as substrates for sequencing on Taq dye deoxy terminator cycle sequencing kits (Applied Biosystem) and fluorescent sequencers (Applied Biosystem Type 370A) according to the included manufacturer's instructions. . As a result, the sequences of nucleotides 32 to 486 of SEQ ID NO: 30 are found. In addition, the results obtained for nucleotides 487 to 991 of SEQ ID NO: 30 correspond to the sequences obtained in section 1.

Mixing with the sequence obtained in section 1 determines the nucleotide sequences 32 to 1950 of SEQ ID NO: 30, which are cDNA nucleotide sequences encoding the megakaryocyte differentiation factor of A431 cells.

In this nucleotide sequence, the translation of all three possible reading frames into an amino acid sequence revealed that one of them had a region that could be translated into a contiguous amino acid sequence containing all of the amino acid sequences shown in SEQ ID NOS: 1-9. And the reading frame of the megakaryocyte differentiation factor was determined.

Within this reading frame is a codon (nucleotides 74-76) for methionine found at the start of translation, from which the region can be translated into an amino acid sequence containing the amino acid sequence shown in SEQ ID NOs: 1-9 It was found that the nucleotide sequence of the nucleotide sequence of 1213 of the nucleotide sequence from position 74 to position 1213 of SEQ ID NO: 30 is the region to be translated into a megakaryocyte differentiation factor.

The nucleotide sequence GCAATGC (nucleotides 71 to 77 of SEQ ID NO: 30) comprising the methionine codons of nucleotides 74 to 76 of SEQ ID NO: 30 is described in literature; M. Kozak, Nucleic Acids Research (1981) Vol. 9, p 5233-5252], corresponding to the sequence (G / A-N-N-A-T-G-G) containing methionine codons that are frequently present at the translation initiation site shown.

Therefore, the primary sequence of the megakaryocyte differentiation factor becomes apparent as 380 amino acids in the structural gene, 42904.43 molecular weight expectation, and 6.79 isoelectric expectation expectation. SEQ ID NO: 1 corresponds to amino acids 188 to 196 of SEQ ID NO: 30; SEQ ID NO: 2 corresponds to amino acids 181 to 187 of SEQ ID NO: 30; SEQ ID NO: 3 corresponds to amino acids 126 to 144 of SEQ ID NO: 30; SEQ ID NO: 4 corresponds to amino acids 325 to 341 of SEQ ID NO: 30; SEQ ID NO: 5 corresponds to amino acids 289 to 297 of SEQ ID NO: 30; SEQ ID NO: 6 corresponds to amino acids 305 to 324 of SEQ ID NO: 30; SEQ ID NO: 7 corresponds to amino acids 121 to 125 of SEQ ID NO: 30; SEQ ID NO: 8 corresponds to amino acids 284 to 288 of SEQ ID NO: 30; SEQ ID NO: 9 corresponds to amino acids 204 to 213 of SEQ ID NO: 30. In addition, the poly A addition signal AATAAA sequence is present at nucleotides 1933 to 1998 of SEQ ID NO: 30.

Example 3

Isolation and Identification of cDNA Encoding Megakaryocyte Differentiation Factor from A431 by PCR and Construction of Expression Vectors

Oligomeric NI078 (SEQ ID NO: 32) and NI079 (SEQ ID NO: 33) are synthesized based on the sequence obtained in Example 2 (SEQ ID NO: 30). In NI078, the sequences of nucleotides 13-37 comprising a translational initiation methionine codon correspond to the sequences of nucleotides 74-98 of SEQ ID NO: 30 and correspond to the EcoRI recognition sites (nucleotides 4-9) and the NruI recognition sites (nucleotides 8-13). Add artificially; In NI079, the sequences of nucleotides 17 to 49 correspond to the nucleotide sequences of nucleotides 1237 to 1269 of SEQ ID NO: 30 and artificially add EcoRI recognition sites (nucleotides 3 to 8) and NotI recognition sites (nucleotides 9 to 16).

Example 2, RNA having poly A prepared from A431 cells in section 1, preamplification system (Gibco BRL) and oligomers, such as random hexamers contained in the system, were prepared using the manufacturer's instructions One strand cDNA was synthesized and PCR synthesized using DNA synthesized as a template and NI078 and NI079 ALC Ampli Taq (Perkin Elmer Cetus) as primers. As a result, a DNA fragment of 1224 bp which is a cDNA fragment for megakaryocyte differentiation factor and has all the information related to megakaryocyte differentiation factor is obtained.

Such DNA fragments are treated with EcoRI to generate EcoRI binding sites at both ends of the cDNA encoding megakaryocyte differentiation factor by EcoRI recognition sites artificially added to oligomers NI078 (SEQ ID NO: 32) and NI079 (SEQ ID NO: 33). This cDNA fragment encoding a megakaryotic differentiation factor is introduced into the mammalian expression vector pdKCR-DHFD at its EcoRI recognition site to yield PdKCR-DHFR-TP055.

Animal cell expression vector PdKCR-dhfr [see; Oikawa, S, et al., Biochem. Biophys. Res. Commun. 164, 39, 1989 refer to pKCRs; 0'Hare et. al., Pro. Natl. Acad. Sci. USA, 78, 1527, 1981 and has a SU 40 early promoter and rabbit β-globin gene and dhfr (dehydroplate reductase) gene. The host transformed with the expression vector was designated Escherichia coli SBM 308, and was deposited on June 7, 1990 by Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 3-chome, Tsukubashi, Ibaraki. , Japan] was deposited with the accession number of FERM P-11506, and on February 18, 1993, it was transferred to the International Deposit under the Treaty of Budapest as FERM BP-4197.

Clone PdKCR-DHFR-TP055 containing megakaryocyte differentiation factor cDNA integrated into pdKCR-DHFR was included in the instructions included using Taq dye deoxy terminator cycle sequencing kit (Applied Biosystem) and fluorescent sequencer (Applied Biosystem Type 370A). To be sequenced accordingly. As a result, the determined nucleotide sequence corresponds to the nucleotides 99 to 1236 of SEQ ID NO: 30 and oligomers NI078 and NI079. Additionally, by sequencing, the megakaryocyte differentiation factor cDNA inserted into the vector is present in the correct orientation for the expression vector promoter. Was confirmed.

As shown above, given the information in SEQ ID NO: 30, for those of ordinary skill in the art, the nucleotide sequence may be expressed as a complete megakaryocyte differentiation factor or any on a megakaryocyte differentiation factor expressing cell line (e.g., A431) and in any expression vector. This can be readily determined by amplifying the cDNA encoding the region.

Example 4

Expression of megakaryocyte differentiation factor in Bombyx mori

(1) Construction of Bombyx mori expression vector

The megakaryocyte differentiation factor cDNA clone pdKCR-DHFR-TP055 is digested with NotI to cleave the NotI recognition site artificially added to NI079. The NotI binding ends thus produced are blunt-terminated using a smoothing kit available from Takara Shuzo, and XbaI linker (Takara Shuzo) is added to the blunt ends according to the attached instructions. The plasmid thus obtained was simultaneously digested with NruI and XbaI simultaneously, and cleaved from the NruI recognition site artificially added to NI078 and the XbI recognition site of the introduced XbaI linker, resulting in megakaryocyte differentiation factor cDAN having an NruI sticky end and an XbaI sticky end. To prepare. This DNA fragment from the baculovirus transfer vector pBm4 for Bombyx mori nuclear polyhedral virus simultaneously digested with NruI and XbaI from the Department of Insect Genetics and Bleeding National Institute of Sericultural and Entomological Science, Ohwashi, Tukuba, Ibaraki 305, JaPan Obtained] is inserted at the NruI recognition site to obtain pBm4-TP055.

(2) Construction of TP055 Recombinant Virus

Cell line BoMo15AIIc derived from Bombyx mori embryonic [Department of Department of Insect Genetics and Bleeding National Institute of Sericultural and Entomological Science, Ohwashi, Tukuba, Ibarki 305, Japan] was extracted from 10% calf serum (FBS: Passage at 25 ° C. in medium containing 500 μg / ml gentamicin in GIBCO BRL) and MGM 448. The TP055 recombinant virus is constructed by co-introducing Bombyx mori nuclear polyhedral virus gene DNA and pBm4-TP055 plasmid DNA into Bombyx mori cultured cells, for example, by calcium phosphate coprecipitation.

That is, 240 μl of sterile purified water of 2 μg of genomic DNA and 10 μg of the transfer plasmid pBm4-TP055 of wild type virus B6E [Department of Insect Genetics and Bleeding National Institute of Sericultural and Entomological Science, Ohwashi, Tukuba, Ibaraki 305, Japan] To this solution, equal volumes of 0.5 M CaCl 2 and 0.1 M HEPES were added, and the mixture was mixed and left at room temperature for 10 minutes. 480 μL of 0.2 M NaCl, 0.05 M HEPES, 0.75 mM NaH 2 PO 4 and 0.75 mM Na 2 HPO 4 are added to the mixture, and the mixture is stirred for several seconds and left at room temperature for 20 to 30 minutes to form a calcium phosphate gel containing genomic DNA and plasmid. .

960 μl of calcium phosphate gel suspension containing viral genomic DNA and transition vector is then added to 4 ml of BoMo15AIIc cells in a 25 cm ² T flask (T25, Corning) and the mixture is left for 12 hours. Replace the medium with fresh MGM 448 (containing 10% FBS and antibiotics) and incubate at 25 ° C. On day 6, the culture medium is recovered as a viral solution.

The culture medium was centrifuged to obtain a clear supernatant, diluted, and then added to BoMo15AIIc cells incubated on a microtiter plate, and after 8 days, a culture medium in which viral infection was observed under a microscope but no polyhedron was selected. (By limiting dilution method). Recover the culture medium. No contamination with wild type virus in virus solution factor was observed.

The recombinant virus constructed as above containing DNA encoding a megakaryotic differentiation factor is designated as TP055-BmNPV.

(3) Expression test of recombinant gene

About 1 × 10 ⁶ BoMo15AIIC cells are incubated for 2 days in plate culture in 4 ml of MGM 448 medium containing 10% FBS in a flask of 25 cm 2 bottom area, in culture the recombinant virus containing the gene encoding a megakaryocyte differentiation factor. (TP055-BmNPV) or 0.5 ml of wild-type virus B6E is added to infect BoMo15AIIc cells, the cells are incubated at 25 ° C. for 3 days, and total RNA is extracted using Isogen (Wako Pure Chemical). Similarly, total RNA is extracted from uninfected BoMo15AIIc cells.

Then, 1 Mg of the RNA thus extracted is size-fractionated by agarose gel electrophoresis, and the separated RNA is transferred to Zetaprobe membrane by capillary action. The membrane is immersed in a hybridization buffer containing the megakaryocyte differentiation factor cDNA (PCR product amplified with KY100 and NI065 described in Example 2.1) (TP055 probe DNA) labeled with Digoxigenin (Boehringer Mannheim) and the mixture is 42 ° C. Incubation was carried out for 12 hours to form a specific complex of recombinant megakaryocyte differentiation factor mRNA and its TP055 probe DNA. The complex is then reacted with an alkaline phosphatase-linked anti-digoxigenin antibody (Boehringer Mannheim) and complexed megakaryocyte differentiation factor mRNA is subjected to luminogen PPD (AMPPD) (Boehringer Mannheim) as alkaline phosphatase according to the manufacturer's instructions. It is detected by chemiluminescence generated by hydrolysis.

As shown in FIG. 11, recombinant megakaryocyte differentiation factor mRMA is detected in total RNA extracted from TP055-BmNPV-infected cells, indicating that mRNA is expressed in TF055-BmNFV-infected cells. On the other hand, expression of mRNA hybridizing with probe DNA is not observed in B6E-infected and uninfected cells.

(4) Solution Preparation of Recombinant Virus

Incubate about 1 × 10 ⁶ BoMo15AIIC cells in 4 ml of MGM448 containing 10% FBS in a bottomed 25 cm ² flask for 2 days, and culture the BoMo15AIIc cells containing the recombinant virus cloned in section 2 above into the vesical fluid. 10 μl of medium is added. After 14 days of incubation at 25 ° C., the culture medium was centrifuged at 1000 rpm for 5 minutes to obtain a supernatant as a recombinant virus solution.

(5) Manufacture of Hemoglobin in Bombyx mori

10 obtained in the section (3) ^-1 and the recombinant virus solution diluted or 10 ^-1 dilution of wild type virus in the virus solution B6E eungaek 50㎕ / head (head), the spring 5 instar larvae injected to Biggs memory, and a commercially available artificial Feed the silkworms (Morus; Katakura Kogyo) at 20 ° C. for 4-5 days. Fifty silkworms were cut to the abdomen, and the extract containing hemolymph and the contents of the central intestine were taken in an ice cooled plastic tube and centrifuged to obtain a supernatant.

(6) Confirmation of activity of megakaryocyte differentiation factor

50 ml of hemolymph of silkworm obtained in section 5 was dialyzed against 20 mM Tris / HCl (pH 7.4) buffer and applied to Matrex Blue A column (ψ 2.5x15 cm) equilibrated with the same buffer. The column is washed thoroughly with the same buffer to remove unbound fractions and the bound protein is eluted with a concentration gradient of 0-1 M NaCl. The elution profile for the megakaryocyte differentiation activity of hemolymph obtained from silkworms injected with recombinant virus is compared with the profile for wild type virus.

As shown in FIG. 12, megakaryocyte differentiation activity in hemolymph from silkworms injected with recombinant virus is much higher than activity against wild type virus.

Bombyx mori baculovirus transfer vectors pBm4, Bombyx mori nuclear polyhedral virus PbE, and Bombix mori cell BoMo15AIIc were used in Example 4, but the present invention is not limited to the use of these materials. That is, using other baculovirus transfer vectors (e.g., pBk283, pBKblue, Funakoshi) Bombyx mori nuclear polyhedron virus (purified DNA available from Funakoshi), Bombyx mori cells (BmN4 cells available from Funakoshi) Thus one skilled in the art can readily obtain megakaryocyte differentiation factor.

The megakaryocyte differentiation factor of the present invention accelerates the formation of megakaryocytes from myeloid cells in the presence of IL-3. The megakaryocyte differentiation factor of the present invention plays an important role in megakaryocyte differentiation and acts in vivo as a precursor. Thus, the megakaryocyte differentiation factor of the present invention may be an effective agent for controlling the reduction of platelet number by radiation or in the case of chemotherapy of tumors as well as in various diseases related to platelet reduction, as well as in the case of bone marrow permeation radiation.

Sequence list

SEQ ID NO: 1

Sequence type: amino acid

Sequence length: 9

Type: Linear

Molecular Type: Peptide

order

SEQ ID NO: 2

Sequence type: amino acid

Sequence length: 7

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 3

Sequence type: amino acid

Sequence length: 19

Type: linear

Molecular Type: Peptide

order

SEQ ID NO: 4

Sequence type: amino acid

Sequence length: 17

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 5

Sequence type: amino acid

Sequence length: 9

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 6

Sequence type: amino acid

Sequence length: 20

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 7

Sequence type: amino acid

Sequence length: 5

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 8

Sequence type: amino acid

Sequence length: 5

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 9

Sequence type: amino acid

Sequence length: 10

Form: Linear

Molecular Type: Peptide

order

SEQ ID NO: 10

Sequence type: nucleic acid

Sequence length: 38

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

Characteristic: Corresponding to amino acid sequence of SEQ ID NO: 3 (I is inosine)

order

SEQ ID NO: 11

Sequence type: nucleic acid

Sequence length: 32

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

Characteristic: Corresponding to amino acid sequence of SEQ ID NO: 4 (I is inosine)

order

SEQ ID NO: 12

Sequence type: nucleic acid

Sequence length: 37

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

Characteristics: Oligomer attached to 3'-RACE kit (Gibco BRL)

order

SEQ ID NO: 13

Sequence type: nucleic acid

Sequence length: 20

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 14

Sequence type: nucleic acid

Sequence length: 23

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 15

Sequence type: nucleic acid

Sequence length: 21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 16

Sequence type: nucleic acid

Sequence length: 21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 17

Sequence type: nucleic acid

Sequence length: 21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 18

Sequence type: nucleic acid

Sequence length '22

Crushing: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 19

Sequence type: nucleic acid

Sequence length: 36

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 20

Sequence type: nucleic acid

Sequence length: 21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 21

Sequence type: nucleic acid

Sequence length: 24

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 22

Sequence type 'nucleic acid

Sequence length: 51

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 23

Sequence type: nucleic acid

Sequence length: 17

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 24

Sequence type: nucleic acid

Sequence length: 17

Main print: Japanese print

Type: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 25

Sequence type: nucleic acid

Sequence length: 20

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 26

Sequence type: nucleic acid

Sequence length: 20

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 27

Sequence type: nucleic acid

Sequence length: 21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 28

Sequence type: nucleic acid

Sequence length '21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA Sequence

SEQ ID NO: 29

Sequence type: nucleic acid

Sequence length: 21

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 30

Sequence type: nucleic acid

Sequence length: 1950

Main print: Double print

Form: Linear

Molecular Type: Complementary DNA (cDNA)

Original source: person

Adjacent source: A431

Characteristics: DNA encoding human megakaryocyte differentiation factor

order

SEQ ID NO: 31

Sequence type: nucleic acid

Sequence length: 20

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

Original source: person

Characteristics: 5'-non-detox area

order

SEQ ID NO: 32

Sequence type: nucleic acid

Sequence length: 37

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

SEQ ID NO: 33

Sequence type: nucleic acid

Sequence length: 49

Main print: Japanese print

Form: Linear

Molecular Type: Synthetic DNA

order

FIG. 1 shows the elution profile of protein (A280- •-) and megakaryocyte differentiation factor (acetylcholine esterase activity-○-) from Q-Sepharose column, where elution is NaCl gradient (0 to 1.0 M) To give fractions 1 to 120.

2 shows the elution profile of protein (A280- •-) and megakaryocyte differentiation factor (acetylcholine esterase activity-○-) from phenyl-sepharose column, where elution is ammonium sulfate concentration gradient (30 to 0). %) And an ethylene glycol concentration gradient (0 to 50%) to yield 1 to 100 fractions, followed by 101 to 120 fractions using 50% ethylene glycol.

Figure 3 shows the elution profile of protein (A280-) and megakaryocyte differentiation factor (acetylcholine esterase activity--) from S-Sepharose column, where elution is NaCl gradient (0 to 0, 5M) to give 1 to 100 fractions.

Figure 4 shows the elution profile of protein (A280-) and megakaryocyte differentiation factor (acetylcholine esterase activity-○-) from Hiroad 26/60 Superdex 75Pg column, obtained by SDS-PAGE The analysis results of the fractions (bottom of the figure) are shown.

5 shows an electrophoretic pattern showing the results of SDS-PAGE analysis of purified megakaryocyte differentiation factor.

FIG. 6 shows the results of isoelectric focusing of the purified megakaryocyte differentiation factor.

FIG. 7 shows an electrophoretic pattern showing the results of sugar chain analysis of megakaryocyte differentiation factor purified by SDS-PAGE, where the first column represents the results of untreated megakaryocyte differentiation factor, and FIG. The column shows the results of the endoglycosidase F-treated megakaryocyte differentiation factor (note; the band near the 35KD position is derived from the enzyme preparation).

8 shows a graph comparing acetylcholine esterase activity of megakaryocytes derived from mouse bone marrow cells cultured for 5 days with or without IL-6 in the presence and absence of purified megakaryocyte differentiation factor (55KDa protein). It is shown.

9 shows the results of acetylcholine esterase staining of mouse bone marrow cells cultured for 4 days after the addition of IL-3, in the presence (B) or absence (A) of purified megakaryocyte differentiation factor.

FIG. 10 shows May-Gruenwald-Giemsa's of mouse bone marrow cells cultured for 4 days after the addition of IL-3, in the presence (B) or absence (A) of purified megakaryocyte differentiation factor. The result of staining is shown.

FIG. 11 shows the detection of RNA using DNA probes (PCR products amplified using KY100 and N1065 as described in Examples 2, 1), wherein the RNA is recombinant containing a gene encoding a megakaryocyte differentiation factor. With cultured Bombyx moli cells (lane B) infected with virus (TF055-Bm NPV), cells infected with wild type virus (B6E) (rail A) or non-transformed cells (lane C) Is extracted from.

FIG. 12 shows a graph showing the expression of megakaryocyte differentiation factor (TP55) in Bombyx mori hemolymph after collection by Matex Blue A column chromatography.

Claims (11)

Produced by cells derived from human epidermal cancer cell A431 cultured in protein-free medium, (1) stimulate the differentiation of megakaryocytes, (2) shows a molecular weight of 55 to 57 kD as measured by gel filtration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and there is no intermolecular disulfide bond, (3) shows an isoelectric point of 6 5 ± 0.5, (4) A megakaryocyte differentiation factor having an amino acid sequence comprising one or more of the amino acid sequences shown in SEQ ID NOs: 1-9 in the sequence list. A megakaryocyte differentiation factor consisting essentially of the amino acid sequence shown in SEQ ID NO: 30. The glycosylated megakaryocyte differentiation factor of claim 2. The megakaryocyte differentiation factor of claim 2, wherein the N-terminus is biochemically or chemically modified. 5. The megakaryocyte differentiation factor of claim 4, wherein the first methionine is deleted and the second alanine is acetylated at the N-terminus. An isolated DNA encoding a megakaryocyte differentiation factor according to claim 2. An expression vector comprising the DNA according to claim 6. A host selected from E. coli and Bombyx mori, transformed with the expression vector according to claim 7. An antibody against the megakaryocyte differentiation factor according to claim 2. A method of producing a megakaryocyte differentiation factor according to claim 2 comprising culturing the host according to claim 8 and recovering the megakaryocyte differentiation factor produced thereby. The method of claim 10, wherein the host is silkworm Bombyx mori.